The goal of this research program is to exploit, and further develop, techniques for manipulating neural activity to identify the brain circuits underlying specific behaviors. Using the Gal4-UAS gene targeting system of Drosophila melanogaster to drive the expression of genes whose products inhibit neuronal excitability, we are selectively suppressing the activity of subsets of neurons and analyzing the effects of this manipulation on behavior. We are particularly interested in the suite of hormonally coordinated and developmentally programmed behaviors executed by the adult fly shortly after emergence from the pupal case, with an immediate focus on those necessary for wing expansion. [unreadable] We have used this approach to identify two functionally distinct groups of neurons which are necessary for wing expansion. Both groups express a common neuropeptide known as Crustacean Cardioactive Peptide (CCAP), but one group (the output group) also expresses and secretes the hormone bursicon into the hemolymph (blood), while the other (the regulatory group) modulates the activity of the output group. Both of these groups consist of multiple neurons, only some of which may be necessary for wing expansion. In addition, individual neurons (or subsets of neurons) within each group may subserve different functional roles. To further determine the functional identities of neurons within the two broad groups we have identified, we have developed a modified version of the Gal4-UAS technique which allows us to selectively manipulate small subsets of CCAP-expressing neurons. [unreadable] Our Split Gal4 system incorporates technology from the yeast two-hybrid system in that it divides the Gal4 molecule into its component DNA-binding (DBD) and transcription activation (TA) domains. We have fused each domain to one of two complementary, heterodimerizing leucine zippers so that the DBD and TA domains associate in cells that express both domains to reconstitute Gal4 transcriptional activity. By independently targeting the two domains in vivo, we can activate UAS transgenes selectively in the subset of cells that expresses both domains. We have exploited this system by targeting the DBD domain to CCAP-expressing neurons and making TA enhancer trap lines that express the Gal4 TA (or the more potent TA of the HSV-1 VP16 transcription factor) in arbitrary patterns that include different subsets of the CCAP-expressing neurons. In a preliminary screen, we have generated lines that permit expression of UAS-transgenes in approximately 38 unique subsets of CCAP-expressing neurons. We are currently analyzing the consequences of ablating these subsets of neurons by tartgeted expression of the cell death gene reaper. Preliminary analysis has already allowed us to identify a critical subset of 16 neurons within the regulatory group. Our analysis further indicates that the 14 neurons of the output group are likely to be functionally redundant. [unreadable] To supplement the available methods for targeted suppression of neuronal activity, we have also been developing tools that permit the selective enhancement or induction of neuronal activity. Just as techniques for suppressing activity can be used to demonstrate which neurons are necessary for a specific behavior, techniques for enhancing activity can be used to demonstrate which neurons are sufficient to drive that behavior. Previously, we have exploited the gene encoding the bacterial sodium channel, NaChBac (fused to Green Fluorescent Protein), to constitutively enhance cellular excitability and have shown that enhanced excitability in the regulatory group of CCAP-expressing neurons disrupts wing expansion and secretion of the hormone bursicon. More recently, we have succeeded in developing the rat cold and menthol receptor (TRPM8) as a tool for acutely activating targeted neurons. We have used this tool to map the critical period for enhancement of excitability in CCAP-expressing neurons to a time window shortly preceding emergence from the pupal case. We are now beginning to acutely activate subsets of CCAP- and bursicon-expressing neurons using TRPM8 in an attempt to identify a minimal subset that might be sufficient to induce the wing expansion program. [unreadable] Investigation of the neuronal substrates of posteclosion behavior in Drosophila using the broad palette of tools we are developing should serve as a proof of concept of a circuit mapping approach that can later be extended to studies of mammalian behavior.